“Some of these stories are closer to my own life than others are, but not one of them is as close as people seem to think.” Alice Murno, from the intro to Moons of Jupiter

"Talent hits a target no one else can hit; genius hits a target no one else can see." Arthur Schopenhauer

“Why does everything you know, and everything you’ve learned, confirm you in what you believed before? Whereas in my case, what I grew up with, and what I thought I believed, is chipped away a little and a little, a fragment then a piece and then a piece more. With every month that passes, the corners are knocked off the certainties of this world: and the next world too. Show me where it says, in the Bible, ‘Purgatory.’ Show me where it says ‘relics, monks, nuns.’ Show me where it says ‘Pope.’” –Thomas Cromwell imagines asking Thomas More—Wolf Hall by Hilary Mantel

Sunday, March 30, 2014

The Better-than-Biblical History of Humanity Hidden in Tiny Cells and a Great Story of Science Hidden in Plain Sight

Anthropology
enthusiasts became acquainted with the name Svante Pääbo in books or articles
published throughout the latter half of this century’s first decade about how
our anatomically modern ancestors might have responded to the presence of other
species of humans as they spread over new continents tens of thousands of years
ago. The first bit of news associated with this unplaceable name was that
humans probably never interbred with Neanderthals, a finding that ran counter
to the multiregionalist theory of human evolution and lent support to the
theory of a single origin in Africa. The significance of the Pääbo team’s
findings in the context of this longstanding debate was a natural enough angle
for science writers to focus on. But what’s shocking in hindsight is that so
little of what was written during those few years conveyed any sense of wonder at
the discovery that DNA from Neanderthals, a species that went extinct 30,000
years ago, was still retrievable—that snatches of it had in fact already been
sequenced.

Then, in 2010, the
verdict suddenly changed; humans really had bred with Neanderthals, and all
people alive today who trace their ancestry to regions outside of Africa carry
vestiges of those couplings in their genomes. The discrepancy between the two
findings, we learned, was owing to the first being based on mitochondrial DNA
and the second on nuclear DNA. Even those anthropology students whose knowledge
of human evolution derived mostly from what can be gleaned from the shapes and
ages of fossil bones probably understood that since several copies of
mitochondrial DNA reside in every cell of a creature’s body, while each cell
houses but a single copy of nuclear DNA, this latest feat of gene sequencing must have been an even greater challenge. Yet, at least among anthropologists,
the accomplishment got swallowed up in the competition between rival scenarios
for how our species came to supplant all the other types of humans. Though, to be
fair, there was a bit of marveling among paleoanthropologists at the
implications of being some percentage Neanderthal.

Fortunately
for us enthusiasts, in his new book Neanderthal Man: In Search of Lost Genomes, Pääbo, a Swedish molecular biologist now
working at the Max Planck Institute in Leipzig, goes some distance toward
making it possible for everyone to appreciate the wonder and magnificence of
his team’s monumental achievements. It would have been a great service to
historians for him to simply recount the series of seemingly insurmountable
obstacles the researchers faced at various stages, along with the technological
advances and bursts of inspiration that saw them through. But what he’s done
instead is pen a deeply personal and sparely stylish paean to the field of
paleogenetics and all the colleagues and supporters who helped him create it.

It’s been over sixty
years since Watson and Crick, with some help from Rosalind Franklin, revealed
the double-helix structure of DNA. But the Human Genome Project, the massive
effort to sequence all three billion base pairs that form the blueprint for a
human, was completed just over ten years ago. As inexorable as the march of
technological progress often seems, the jump from methods for sequencing the
genes of living creatures to those of long-extinct species only strikes us as
foregone in hindsight. At the time when Pääbo was originally dreaming of
ancient DNA, which he first hoped to retrieve from Egyptian mummies, there were
plenty of reasons to doubt it was possible. He writes,

When
we die, we stop breathing; the cells in our body then run out of oxygen, and as
a consequence their energy runs out. This stops the repair of DNA, and various
sorts of damage rapidly accumulate. In addition to the spontaneous chemical
damage that continually occurs in living cells, there are forms of damage that
occur after death, once the cells start to decompose. One of the crucial functions
of living cells is to maintain compartments where enzymes and other substances
are kept separate from one another. Some of these compartments contain enzymes
that break down DNA from various microorganisms that the cell may encounter and
engulf. Once an organism dies and runs out of energy, the compartment membranes
deteriorate, and these enzymes leak out and begin degrading DNA in an
uncontrolled way. Within hours and sometimes days after death, the DNA strands
in our body are cut into smaller and smaller pieces, while various other forms
of damage accumulate. At the same time, bacteria that live in our intestines
and lungs start growing uncontrollably when our body fails to maintain the
barriers that normally contain them. Together these processes will eventually
dissolve the genetic information stored in our DNA—the information that once
allowed our body to form, be maintained, and function. When that process is
complete, the last trace of our biological uniqueness is gone. In a sense, our
physical death is then complete. (6)

The hope was that amid this nucleic carnage enough
pieces would survive to restore a single strand of the entire genome. That
meant Pääbo needed lots of organic remains and some really powerful extraction
tools. It also meant that he’d need some well-tested and highly reliable
methods for fitting the pieces of the puzzle together.

Along
with the sense of inevitability that follows fast on the heels of any
scientific advance, the impact of the Neanderthal Genome Project’s success in
the wider culture was also dampened by a troubling inability on the part of the
masses to appreciate that not all ideas are created equal—that any particular theory is only as good as the path researchers followed to arrive at it and the
methods they used to validate it. Sadly, it’s in all probability the very
people who would have been the most thoroughly gobsmacked by the findings
coming out of the Max Planck Institute whose amazement switches are most susceptible
to hijacking at the hands of the charlatans and ratings whores behind shows
like Ancient Aliens. More serious
than the cheap fictions masquerading as science that abound in pop culture,
though, is a school of thought in academia that not only fails to grasp, but
outright denies, the value of methodological rigor, charging that the methods
themselves are mere vessels for the dissemination of encrypted social and
political prejudices.

Such thinking can’t
survive even the most casual encounter with the realities of how science is
conducted. Pääbo, for instance, describes his team’s frustration whenever rival
researchers published findings based on protocols that failed to meet the
standards they’d developed to rule out contamination from other sources of
genetic material. He explains the common “dilemma in science” whereby

doing
all the analyses and experiments necessary to tell the complete story leaves
you vulnerable to being beaten to the press by those willing to publish a less complete
story that nevertheless makes the major point you wanted to make. Even when you
publish a better paper, you are seen as mopping up the details after someone
who made the real breakthrough. (115)

The more serious challenge for Pääbo, however, was
dialing back extravagant expectations on the part of prospective funders against the backdrop of popular notions propagated by the Jurassic
Park movie franchise and extraordinary claims from scientists who should’ve
known better. He writes,

As we
were painstakingly developing methods to detect and eliminate contamination, we
were frustrated by flashy publications in Nature
and Science whose authors, on the
surface of things, were much more successful than we were and whose accomplishments
dwarfed the scant products of our cumbersome efforts to retrieve DNA sequences
“only” a few tens of thousands of years old. The trend had begun in 1990, when
I was still at Berkeley. Scientists at UC Irvine published a DNA sequence from
leaves of Magnolia latahensis that
had been found in a Miocene deposit in Clarkia, Idaho, and were 17 million
years old. This was a breathtaking achievement, seeming to suggest that one
could study DNA evolution on a time scale of millions of years, perhaps even
going back to the dinosaurs! (56)

In
the tradition of the best scientists, Pääbo didn’t simply retreat to his own
projects to await the inevitable retractions and failed replications but
instead set out to apply his own more meticulous extraction methods to the
fossilized plant material. He writes,

I
collected many of these leaves and brought them back with me to Munich. In my
new lab, I tried extracting DNA from the leaves and found they contained many
long DNA fragments. But I could amplify no plant DNA by PCR. Suspecting that
the long DNA was from bacteria, I tried primers for bacterial DNA instead, and
was immediately successful. Obviously, bacteria had been growing in the clay.
The only reasonable explanation was that the Irvine group, who worked on plant
genes and did not use a separate “clean lab” for their ancient work, had
amplified some contaminating DNA and thought it came from the fossil leaves.
(57)

With the right equipment, it turns out, you can
extract and sequence genetic material from pretty much any kind of organic
remains, no matter how old. The problem is that sources of contamination are
myriad, and chances are whatever DNA you manage to read is almost sure to be
from something other than the ancient creature you’re interested in.

At
the time when Pääbo was busy honing his techniques, many scientists thought
genetic material from ancient plants and insects might be preserved in the
fossilized tree resin known as amber. Sure enough, in the late 80s and early 90s,
George Poinar and
Raul Cano published a series of articles in which they claimed to have successfully
extracted DNA through tiny holes drilled into chunks of amber to reach embedded
bugs and leaves. These articles were in fact the inspiration behind Michael
Crichton’s description of how the dinosaurs in Jurassic Park were cloned. But Pääbo had doubts about whether these
researchers were taking proper precautions to rule out contamination, and no
sooner had he heard about their findings than he started trying to find a way
to get his hands on some amber specimens. He writes,

The
opportunity to find out came in 1994, when Hendrik Poinar joined our lab.
Hendrik was a jovial Californian and the son of George Poinar, then a
professor at Berkeley and a well-respected expert on amber and the creatures
found in it. Hendrik had published some of the amber DNA sequences with Raul
Cano, and his father had access to the best amber in the world. Hendrik came to
Munich and went to work in our new clean room. But he could not repeat what had
been done in San Luis Obisco. In fact, as long as his blank extracts were
clean, he got no DNA sequences at all out of the amber—regardless of whether he
tried insects or plants. I grew more and more skeptical, and I was in good
company. (58)

Those blank extracts were important not just to
test for bacteria in the samples but to check for human cells as well. Indeed,
one of the special challenges of isolating Neanderthal DNA is that it looks so
much like the DNA of the anatomically modern humans handling the samples and
the sequencing machines.

A high percentage of the
dust that accumulates in houses is made up of our sloughed off skin cells. And
Polymerase Chain Reaction (PCR), the technique Pääbo’s team was
using to increase the amount of target DNA, relies on a powerful amplification process which uses rapid heating and
cooling to split double helix strands up the middle before fitting synthetic
chemicals along each side like an amino acid zipper, resulting in exponential
replication. The result is that each fragment of a genome gets blown up, and it
becomes impossible to tell what percentage of the specimen’s DNA it originally
represented. Researchers then try to fit the fragments end-to-end based on
repeating overlaps until they have an entire strand. If there’s a great deal of
similarity between the individual you’re trying to sequence and the individual
whose cells have contaminated the sample, you simply have no way to know
whether you’re splicing together fragments of each individual’s genome. Much of
the early work Pääbo did was with extinct mammals like giant ground sloths
which were easier to disentangle from humans. These early studies were what led
to the development of practices like running blank extracts, which would later
help his team ensure that their supposed Neanderthal DNA wasn’t really from
modern human dust.

Despite all the claims of
million-year-old DNA being publicized, Pääbo and his team eventually had to
rein in their frustration and stop “playing the PCR police” (61) if they ever
wanted to show their techniques could be applied to an ancient species of
human. One of the major events in Pääbo’s life that would make this huge
accomplishment a reality was the founding of the Max Planck Institute for
Evolutionary Anthropology in 1997. As celebrated as the Max Planck Society is today, though, the idea
of an institute devoted to scientific anthropology in Germany at the time had
to overcome some resistance arising out of fears that history might repeat
itself. Pääbo explains,

As do
many contemporary German institutions, the MPS had a predecessor before the
war. Its name was the Kaiser Wilhelm Society, and it was founded in 1911. The
Kaiser Wilhelm Society had built up and supported institutes around eminent
scientists such as Otto Hahn, Albert Einstein, Max Planck, and Werner
Heisenberg, scientific giants active at a time when Germany was a
scientifically dominant nation. That era came to an abrupt end when Hitler rose
to power and the Nazis ousted many of the best scientists because they were
Jewish. Although formally independent of the government, the Kaiser Wilhelm
Society became part of the German war machine—doing, for example, weapons
research. This was not surprising. Even worse was that through its Institute
for Anthropology, Human Heredity, and Eugenics the Kaiser Wilhelm Society was
actively involved in racial science and the crimes that grew out of that. In
that institute, based in Berlin, people like Josef Mengele were scientific
assistants while performing on inmates at Auschwitz death camp, many of them children. (81-2)

Even without such direct historical connections,
many scholars still automatically leap from any mention of anthropology or
genetics to dubious efforts to give the imprimatur of science to racial
hierarchies and clear the way for atrocities like eugenic culling or sterilizations,
even though no scientist in any field would have truck with such ideas and
policies after the lessons of the past century.

Pääbo
not only believed that anthropological science could be conducted without
repeating the atrocities of the past; he insisted that allowing history to rule
real science out of bounds would effectively defeat the purpose of the entire
endeavor of establishing an organization for the study of human origins. Called
on as a consultant to help steer a course for the institute he was
simultaneously being recruited to work for, Pääbo recalls responding to the
administrators’ historical concerns,

Perhaps
it was easier for me as a non-German born well after the war to have a relaxed
attitude toward this. I felt that more than fifty years after the war, Germany
could not allow itself to be inhibited in its scientific endeavors by its past
crimes. We should neither forget history nor fail to learn from it, but we
should also not be afraid to go forward. I think I even said that fifty years
after his death, Hitler should not be allowed to dictate what we could or could
not do. I stressed that in my opinion any new institute devoted to anthropology
should not be a place where one philosophized about human history. It should do
empirical science. Scientists who were to work there should collect real hard
facts about human history and test their ideas against them. (82-3)

As it turned out, Pääbo wasn’t alone in his
convictions, and his vision of what the institute should be and how it should
operate came to fruition with the construction of the research facility in
Leipzig.

Faced
with Pääbo’s passionate enthusiasm, some may worry that he’s one of those mad
scientists we know about from movies and books, willing to push ahead with his
obsessions regardless of the moral implications or the societal impacts. But in
fact Pääbo goes a long way toward showing that the popular conception of the socially
oblivious scientist who calculates but can’t think, and who solves puzzles but
is baffled by human emotions is not just a caricature but a malicious fiction.
For instance, even amid the excitement of his team’s discovery that humans
reproduced with Neanderthals, Pääbo was keenly aware that his results revealed
stark genetic differences between Africans, who have no Neanderthal DNA, and
non-Africans, most of whose genomes are between one and four percent
Neanderthal. He writes,

When
we had come this far in our analyses, I began to worry about what the social
implications of our findings might be. Of course, scientists need to
communicate the truth to the public, but I feel that they should do so in ways
that minimize the chance for it to be misused. This is especially the case when
it comes to human history and human genetic variation, when we need to ask
ourselves: Do our findings feed into prejudices that exist in society? Can our
findings be misrepresented to serve racists’ purposes? Can they be deliberately
or unintentionally misused in some other way? (199-200)

In light of the Neanderthal’s own
caricature—hunched, brutish, dimwitted—their contribution to non-Africans’
genetic makeup may actually seem like more of a drawback than a basis for any
claims of superiority. The trouble would come, however, if some of these genes
turned out to confer adaptive advantages that made their persistence in our
lineage more likely. There are already some indications, for instance, that
Neanderthal-human hybrids had more robust immune responses to certain diseases.
And the potential for further discoveries along these lines is limitless.

Neanderthal Man explores the personal
and political dimensions of a major scientific undertaking, but it’s Pääbo’s
remembrances of what it was like to work with the other members of his team
that bring us closest to the essence of what science is—or at least what it can
be. At several points along the team’s journey, they were faced with a series
of setbacks and technical challenges that threatened to sink the entire
endeavor. Pääbo describes what it was like when during one critical juncture
where things looked especially dire everyone brought their heads together in
weekly meetings to try to come up with solutions and assign tasks:

To me,
these meetings were absorbing social and intellectual experiences: graduate
students and postdocs know that their careers depend on the results they
achieve and the papers they publish, so there is always a certain amount of
jockeying for opportunity to do the key experiments and to avoid doing those
that may serve the group’s aim but will probably not result in prominent
authorship on an important publication. I had become used to the idea that
budding scientists were largely driven by self-interest, and I recognized that
my function was to strike a balance between what was good for someone’s career
and what was necessary for a project, weighing individual abilities in this
regard. As the Neanderthal crisis loomed over the group, however, I was amazed
to see how readily the self-centered dynamic gave way to a more group-centered
one. The group was functioning as a unit, with everyone eagerly volunteering
for thankless and laborious chores that would advance the project regardless of
whether such chores would bring any personal glory. There was a strong sense of
common purpose in what all felt was a historic endeavor. I felt we had the
perfect team. In my more sentimental moments, I felt love for each and every
person around the table. This made the feeling that we’d achieved no progress
all the more bitter. (146-7)

Those “more sentimental moments” of Pääbo’s occur
quite frequently, and he just as frequently describes his colleagues, and even
his rivals, in a way that reveals his fondness and admiration for them. Unlike
James Watson, who in The Double Helix,
his memoir of how he and Francis Crick discovered the underlying structure of
DNA, often comes across as nasty and condescending, Pääbo reveals himself to be
bighearted, almost to a fault.

Alongside
the passion and the drive, we see Pääbo again and again pausing to reflect with childlike wonder at the dizzying advancement of technology and the incredible
privilege of being able to carry on such a transformative tradition of
discovery and human progress. He shows at once the humility of recognizing his
own limitations and the restless curiosity that propels him onward in spite of
them. He writes,

My
twenty-five years in molecular biology had essentially been a continuous
technical revolution. I had seen DNA sequencing machines come on the market
that rendered into an overnight task the toils that took me days and weeks as a
graduate student. I had seen cumbersome cloning of DNA in bacteria be replaced
by the PCR, which in hours achieved what had earlier taken weeks or months to
do. Perhaps that was what had led me to think that within a year or two we
would be able to sequence three thousand times more DNA than what we had
presented in the proof-of-principle paper in Nature. Then again, why wouldn’t the technological revolution
continue? I had learned over the years that unless a person was very, very
smart, breakthroughs were best sought when coupled to big improvements in
technologies. But that didn’t mean we were simply prisoners awaiting rescue by
the next technical revolution. (143)

Like the other members of his team, and like so
many other giants in the history of science, Pääbo demonstrates an important and rare mix of
seemingly contradictory traits: a capacity for dogged, often mind-numbing
meticulousness and a proclivity toward boundless flights of imagination.

Denisovan Finger Bone

What has been the impact
of Pääbo and his team’s accomplishments so far? Their methods
have already been applied to the remains of a 400,000-year-old human
ancestor, led to the discovery of completely new species of hominin known as
Denisovans (based on a tiny finger bone), and are helping settle a longstanding
debate about the peopling
of the Americas. The out-of-Africa hypothesis is, for now, the clear victor
over the multiregionalist hypothesis, but of course the single origin theory
has become more complicated. Many paleoanthropologists are now talking about
what Pääbo calls the “Leaky replacement” model (248). Aside from filling in
some of the many gaps in the chronology of humankind’s origins and
migrations—or rather fitting together more pieces in the vast mosaic of our
species’ history—every new genome helps us to triangulate possible functions
for specific genes. As Pääbo explains, “The dirty little secret of genomics is
that we still know next to nothing about how a genome translates into the
particularities of a living and breathing individual” (208). But knowing the
particulars of how human genomes differ from chimp genomes, and how both differ
from the genomes of Neanderthals, or Denisovans, or any number of living or
extinct species of primates, gives us clues about how those differences
contribute to making each of us who and what we are. The Neanderthal genome is
not an end-point but rather a link in a chain of discoveries. Nonetheless, we
owe Svante Pääbo a debt of gratitude for helping us to appreciate what all went
into the forging of this particular, particularly extraordinary link.

3 comments:

Very accessible and well-written piece. It seems though that books such as this and Dawkins multi-part memoir can only be for hard-core science aficionados. Although ancient DNA is what everyone's talking about right now.

To read the review it seems, and I don't get why, he's intent on dispelling the idea of resurrecting dinosaurs--or is it just dinosaurs from amber. Haven't we already extracted proteins from a dino egg in China? Does Paabo say the theoretical age limit on extracting DNA from fossils? Or is it like you said "no matter how old?"

You brought up a great point that Paabo doesn't seem to address--focusing instead on the safer issue of history--the implication about human variation across populations and adaptive advantages shared by some but not all, once that sinks in to the collective conscious.

I hesitate because it's nit-picky, but PCR is not a "sequencing technique." PCR is one of the most powerful tools ever invented, invented by a surfer on acid.

Sequencing can be broken into three general stages: sample library preparation, sequencing the samples, analyzing the data. PCR is one step of many in the first stage. The PCR cartoon you have is curiously missing an important component of the reaction: the building blocks for all that exponential growth, the four free-floating deoxynucleoside triphosphates.

In whole genome sequencing, PCR is typically used to enrich/increase minuscule amounts of starting DNA after or concurrently during DNA fragment barcoding thus avoiding researcher contamination, but before sequencing and also as a quality control step to see how much "sequenceable" DNA is in your final product or DNA library. The novel method Paabo et al., developed is not in the actual sequencing itself but in that initial prep, which yielded something like 30x coverage (low) with less than 1% contamination from modern humans when mapped back to the *female reference genome. I should read it again but the 2012 Science paper is unclear on how this single-stranded method affords better AT representation in degraded GC biased DNA but whatever.

Nit-pick away. I had anticipated running my dilettantish description by you before posting it, but I was in a rush to get the post up before the end of the month. I've made an edit to correct the error. One of the thrilling/frustrating things about Paabo's writing is that he (necessarily) bounces back-and-forth between technical descriptions and aspects of the narrative, so you're left wanting a bit more of each. The paragraph about PCR was meant to give a taste of the endless technical problems the team faced. It originated with this passage:

"I also found that when there few or even no molecules long enough for the DNA polymerase to operate continuously from one primer to the other,the polymerase would sometimes stitch shorter pieces of DNA together, producing Frankenstein's monster-like combinations that did not exist in the original genome of the ancient organism." (45)

I guess some researchers do this on purpose now to create novel sequences.

Regarding dino-DNA, I didn't get the impression Paabo was against trying to retrieve it--though he was pretty skeptical about DNA sticking around that long. Working in the 90s and early 2000s, though, he was competing for support against people claiming they could, and were about to sequence dino-genes. So it's understandable that he'd be frustrated.

As for variation and adaptive advantages, I haven't really come across any good ways to discuss that issue. Unfortunately, it gets swallowed up in political correctness. The out most writers like to use is referencing the bottleneck in human history, which made us very genetically homogeneous as a species. But aside from the technical difficulties of identifying "warrior genes" or "math genes", you can count on being labeled a Nazi if you even take the possibility seriously.

I'm sure he knows what he's talking about but there is no "long-enough" limit on the length of molecules that can be amplified using primers--at some point you'll be essentially ligating the forward and reverse primers together. What he's probably talking about is the result of shortening the primer from the standard 20 base pairs length. The shorter the primer sequence the greater the chances it will hybridize to a non-specific DNA template. That could produce a spurious product.

the out most writers like to use is referencing the bottleneck in human history

The most obvious counter to charges of Nazism to me is trade-offs and context in evolution. For instance, sickle cell is not universally advantageous, only in certain geographies. Same goes for skin pigment and lactose tolerance.

But we know why those examples are much less controversial, right? It's because those pesky technical difficulties have been mostly hammered out. Besides, context and trade-offs, I think there is a total lack of appreciation for developmental timing in discussions about the expression of behavioral traits with a heritable component. Well that and an under-appreciation for the knowledge gap between genotype and phenotype.

The funny thing about warrior genes (MAO) is they're located on the X chromosome, meaning men only have one copy so they're at higher risk (more heritable) when a mutation is present. Almost half of us have the mutation correlated with aggression but that somehow doesn't lead to half of males with a rap sheet: environmental context and developmental timing.